Cancer diagnostic method based upon DNA methylation differences

ABSTRACT

There is disclosed a cancer diagnostic method based upon DNA methylation differences at specific CpG sites. Specifically, the inventive method provides for a bisulfite treatment of DNA, followed by methylation-sensitive single nucleotide primer extension (Ms-SNuPE), for determination of strand-specific methylation status at cytosine residues.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims priority from U.S. Provisional PatentApplication No. 60/049,231 filed Jun. 9, 1997.

TECHNICAL FIELD OF THE INVENTION

The present invention provides a cancer diagnostic method based upon DNAmethylation differences at specific CpG sites. Specifically, theinventive method provides for a bisulfite treatment of DNA, followed bymethylation-sensitive single nucleotide primer extension (Ms-SNuPE), fordetermination of strand-specific methylation status at cytosineresidues.

BACKGROUND OF THE INVENTION

Cancer treatments, in general, have a higher rate of success if thecancer is diagnosed early and treatment is started earlier in thedisease process. The relationship between improved prognosis and stageof disease at diagnosis hold across all forms of cancer for the mostpart. Therefore, there is an important need to develop early assays ofgeneral tumorigenesis through marker assays that measure generaltumorigenesis without regard to the tissue source or cell type that isthe source of a primary tumor. Moreover, there is a need to addressdistinct genetic alteration patterns that can serve as a platformassociated with general tumorigenesis for early detection and prognosticmonitoring of many forms of cancer.

Importance of DNA Methylation

DNA methylation is a mechanism for changing the base sequence of DNAwithout altering its coding function. DNA methylation is a heritable,reversible and epigenetic change. Yet, DNA methylation has the potentialto alter gene expression, which has profound developmental and geneticconsequences. The methylation reaction involves flipping a targetcytosine out of an intact double helix to allow the transfer of a methylgroup from S-adenosylmethionine in a cleft of the enzyme DNA(cystosine-5)-methyltransferase (Klimasauskas et al., Cell 76:357-369,1994) to form 5-methylcytosine (5-mCyt). This enzymatic conversion isthe only epigenetic modification of DNA known to exist in vertebratesand is essential for normal embryonic development (Bird, Cell 70:5-8,1992; Laird and Jaenisch, Human Mol. Genet. 3:1487-1495, 1994; andBestor and Jaenisch, Cell 69:915-926, 1992). The presence of 5-mCyt atCpG dinucleotides has resulted in a 5-fold depletion of this sequence inthe genome during vertebrate evolution, presumably due to spontaneousdeamination of 5-mCyt to T (Schoreret et al., Proc. Natl. Acad. Sci. USA89:957-961, 1992). Those areas of the genome that do not show suchsuppression are referred to as “CpG islands” (Bird, Nature 321:209-213,1986; and Gardiner-Garden et al., J. Mol. Biol. 196:261-282, 1987).These CpG island regions comprise about 1% of vertebrate genomes andalso account for about 15% of the total number of CpG dinucleotides(Bird, Infra.). CpG islands are typically between 0.2 to about 1 kb inlength and are located upstream of many housekeeping and tissue-specificgenes, but may also extend into gene coding regions. Therefore, it isthe methylation of cytosine residues within CpG islands in somatictissues, which is believed to affect gene function by alteringtranscription (Cedar, Cell 53:3-4, 1988).

Methylation of cytosine residues contained within CpG islands of certaingenes has been inversely correlated with gene activity. This could leadto decreased gene expression by a variety of mechanisms including, forexample, disruption of local chromatin structure, inhibition oftranscription factor-DNA binding, or by recruitment of proteins whichinteract specifically with methylated sequences indirectly preventingtranscription factor binding. In other words, there are several theoriesas to how methylation affects mRNA transcription and gene expression,but the exact mechanism of action is not well understood. Some studieshave demonstrated an inverse correlation between methylation of CpGislands and gene expression, however, most CpG islands on autosomalgenes remain unmethylated in the germline and methylation of theseislands is usually independent of gene expression. Tissue-specific genesare usually unmethylated and the receptive target organs but aremethylated in the germline and in non-expressing adult tissues. CpGislands of constitutively-expressed housekeeping genes are normallyunmethylated in the germline and in somatic tissues.

Abnormal methylation of CpG islands associated with tumor suppressorgenes may also cause decreased gene expression. Increased methylation ofsuch regions may lead to progressive reduction of normal gene expressionresulting in the selection of a population of cells having a selectivegrowth advantage (i.e., a malignancy).

It is considered that altered DNA methylation patterns, particularlymethylation of cytosine residues, cause genome instability and aremutagenic. This, presumably, has led to an 80% suppression of a CpGmethyl acceptor site in eukaryotic organisms, which methylate theirgenomes. Cytosine methylation further contributes to generation ofpolymorphism and germ-line mutations and to transition mutations thatinactivate tumor-suppressor genes (Jones, Cancer Res. 56:2463-2467,1996). Methylation is also required for embryonic development of mammals(Bestor and Jaenisch, Cell 69:915-926, 1992). It appears that that themethylation of CpG-rich promoter regions may be blocking transcriptionalactivity. Therefore, there is a probability that alterations ofmethylation are an important epigenetic criteria and can play a role incarcinogenesis in general due to its function of regulating geneexpression. Ushijima et al. (Proc. Natl. Acad. Sci. USA 94:2284-2289,1997) characterized and cloned DNA fragments that show methylationchanges during murine hepatocarcinogenesis. Data from a group of studiesof altered methylation sites in cancer cells show that it is not simplythe overall levels of DNA methylation that are altered in cancer, butchanges in the distribution of methyl groups.

These studies suggest that methylation, at CpG-rich sequences known asCpG islands, provide an alternative pathway for the inactivation oftumor suppressors, despite the fact that the supporting studies haveanalyzed only a few restriction enzyme sites without much knowledge asto their relevance to gene control. These reports suggest thatmethylation of CpG oligonucleotides in the promoters of tumor suppressorgenes can lead to their inactivation. Other studies provide data thatsuggest that alterations in the normal methylation process areassociated with genomic instability (Lengauer et al. Proc. Natl. Acad.Sci. USA 94:2545-2550, 1997). Such abnormal epigenetic changes may befound in many types of cancer and can, therefore, serve as potentialmarkets for oncogenic transformation, provided that there is a reliablemeans for rapidly determining such epigenetic changes. The presentinvention was made to provide such a universal means for determiningabnormal epigenetic changes and address this need in the art.

Methods to Determine DNA Methylation

There is a variety of genome scanning methods that have been used toidentify altered methylation sites in cancer cells. For example, onemethod involves restriction landmark genomic scanning (Kawai et al.,Mol. Cell. Biol. 14:7421-7427, 1994), and another example involvesmethylation-sensitive arbitrarily primed PCR (Gonzalgo et al., CancerRes. 57:594-599, 1997). Changes in methylation patterns at specific CpGsites have been monitored by digestion of genomic DNA withmethylation-sensitive restriction enzymes followed by Southern analysisof the regions of interest (digestion-Southern method). Thedigestion-Southern method is a straightforward method but it hasinherent disadvantages in that it requires a large amount of DNA (atleast or greater than 5 μg) and has a limited scope for analysis of CpGsites (as determined by the presence of recognition sites formethylation-sensitive restriction enzymes). Another method for analyzingchanges in methylation patterns involves a PCR-based process thatinvolves digestion of genomic DNA with methylation-sensitive restrictionenzymes prior to PCR amplification (Singer-Sam et al., Nucl. Acids Res.18:687,1990). However, this method has not been shown effective becauseof a high degree of false positive signals (methylation present) due toinefficient enzyme digestion of overamplification in a subsequent PCRreaction.

Genomic sequencing has been simplified for analysis of DNA methylationpatterns and 5-methylcytosine distribution by using bisulfite treatment(Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992).Bisulfite treatment of DNA distinguishes methylated from unmethylatedcytosines, but original bisulfite genomic sequencing requireslarge-scale sequencing of multiple plasmid clones to determine overallmethylation patterns, which prevents this technique from beingcommercially useful for determining methylation patterns in any type ofa routine diagnostic assay.

In addition, other techniques have been reported which utilize bisulfitetreatment of DNA as a starting point for methylation analysis. Theseinclude methylation-specific PCR (MSP) (Herman et al. Proc. Natl. Acad.Sci. USA 93:9821-9826, 1992); and restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA (Sadri and Hornsby,Nucl. Acids Res. 24:5058-5059, 1996; and Xiong and Laird, Nucl. Acids.Res. 25:2532-2534, 1997).

PCR techniques have been developed for detection of gene mutations(Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147, 1991) andquantitation of allelic-specific expression (Szabo and Mann, Genes Dev.9:3097-3108, 1995; and Singer-Sam et al., PCR Methods Appl. 1:160-163,1992). Such techniques use internal primers, which anneal to aPCR-generated template and terminate immediately 5′ of the singlenucleotide to be assayed. However an allelic-specific expressiontechnique has not been tried within the context of assaying for DNAmethylation patterns.

Therefore, there is a need in the art to develop improved diagnosticassays for early detection of cancer using reliable and reproduciblemethods for determining DNA methylation patterns that can be performedusing familiar procedures suitable for widespread use. This inventionwas made to address the foregoing need.

SUMMARY OF THE INVENTION

The present invention provides a method for determining DNA methylationpatterns at cytosine sites, comprising the steps of:

(a) obtaining genomic DNA from a DNA sample to be assayed;

(b) reacting the genomic DNA with sodium bisulfite to convertunmethylated cytosine residues to uracil residues while leaving any5-methylcytosine residues unchanged to provide primers specific for thebisulfite-converted genomic sample for top strand or bottom strandmethylation analysis;

(c) performing a PCR amplification procedure using the top strand orbottom strand specific primers;

(d) isolating the PCR amplification products;

(e) performing a primer extension reaction using Ms-SNuPE primers,[³²P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprisefrom about a 15 mer to about a 22 mer length primer that terminatesimmediately 5′ of a single nucleotide to be assayed; and

(f) determining the relative amount of methylation at CpG sites bymeasuring the incorporation of different ³²P-labeled dNTPs.

Preferably, the [³²P]NTP for top strand analysis is [³²P]dCTP or[³²P]TTP. Preferably, the [³²P]NTP for bottom strand analysis is[³²P]dATP or [³²P]dGTP. Preferably, the isolation step of the PCRproducts uses an electrophoresis technique. Most preferably, theelectrophoresis technique uses an agarose gel. Preferably, the Ms-SNuPEprimer sequence comprises a sequence of at least fifteen but no morethan twenty five, bases having a sequence selected from the groupconsisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ ID NO. 2], GaL4 [SEQ ID NO.3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6], HuN3 [SEQ ID NO. 7], HuN4[SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQ ID NO. 9], CaS1 [SEQ IDNO. 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO. 12], and combinationsthereof.

The present invention further provides a Ms-SNuPE primer sequencedesigned to anneal to and terminate immediately 5′ of a desired cytosinecodon in the CpG target site and that is located 5′ upstream from a CpGisland and are frequently hypermethylated in promoter regions of somaticgenes in malignant tissue. Preferably, the Ms-SNuPE primer sequencecomprises a sequence of at least fifteen bases having a sequenceselected from the group consisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ IDNO. 2], GaL4 [SEQ ID NO. 3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6],HuN3 [SEQ ID NO. 7], HuN4 [SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQID NO. 9], CaS1 [SEQ ID NO. 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO.12], and combinations thereof. The present invention further provides amethod for obtaining a Ms-SNuPE primer sequence, comprising finding ahypermethylated CpG island in a somatic gene from a malignant tissue orcell culture, determining the sequence located immediately 5′ upstreamfrom the hypermethylated CpG island, and isolating a 15 to 25 mersequence 5′ upstream from the hypermethylated CpG island for use as aMs-SNuPE primer. The present invention further provides a Ms-SNuPEprimer comprising a 15 to 25 mer oligonucleotide sequence obtained bythe process comprising, finding a hypermethylated CpG island in asomatic gene from a malignant tissue or cell culture, determining thesequence located immediately 5′ upstream from the hypermethylated CpGisland, and isolating a 15 to 25 mer sequence 5′ upstream from thehypermethylated CpG island for use as a Ms-SNuPE primer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the inventive Ms-SNuPE assay for determinationof strand-specific methylation status at cytosines. The process involvestreating genomic DNA with sodium bisulfite, and generating a template bya PCR technique for a top strand methylation analysis. Alternatively abottom strand methylation can also be assayed by designing theappropriate primers to generate a bottom strand-specific template. Theprocess further entails amplifying the templates by a PCR technique. ThePCR products are electrophoresed and isolated from agarose gels,followed by incubation with Ms-SNuPE primers, as disclosed hereinwherein the Ms-SNuPE primers comprise a from about a 15 mer to about a25 mer length primer that terminates immediately 5′ of a singlenucleotide to be assayed, and PCR buffer, [³²P]dNTPs and Taq polymerasefor primer extension reactions. The radiolabeled products are separated,for example, by electrophoresis on polyacrylamide gels under denaturingconditions and visualized by exposure to autoradiographic film orphosphorimage quantitation.

FIG. 2 shows the results from a quantitative methylation analysis ofthree top strand CpG sites from a 5′ CpG island of p16. P16 is a knowntumor suppressor gene and the particular region examined for changes inmethylation is the promoter region of this gene. The top panel providesthe locations of three sites analyzed (numbered 1, 2 and 3) relative tothe putative transcriptional start sites (vertical arrows pointingupwards) and the exon 1α coding domain. The PCR primers used for topstrand amplification of the 5′ region of p16 (which includes putativetranscriptional start sites) were 5′-GTA GGT GGG GAG GAG TTT AGT T-3′[SEQ ID NO. 13] and 5′-TCT AAT AAC CAA CCA ACC CCT CC-3′ [SEQ ID NO.14]. The control sets included “M” PCR product amplified from a plasmidcontaining bisulfide-specific methylated sequence; “U” PCR productamplified from a plasmid containing bisulfite-specific unmethylatedsequence; and “mix” a 50:50 mixture of methylated and unmethylatedPCR-amplified plasmid sequences. The DNA samples analyzed included T24and J82 bladder cancer cell lines; wbc (white blood cell), melanoma(primary melanoma tumor tissue sample), and bladder (primary bladdertumor tissue sample). The tissue samples were micro dissected fromparaffin-embedded tumor material. The grid at the bottom of the lowerpanel shows the ratio of methylated (C) versus unmethylated (T) bands ateach site based upon phosphorimage quantitation.

FIG. 3 shows a mixing experiment showing a linear response of theinventive Ms-SNuPE assay for detection of cytosine methylation. A T24bladder cancer cell line DNA (predominantly methylated) was added inincreasing amounts to a J82 bladder cancer cell line DNA (predominantlyunmethylated). FIG. 3 shows data from an 18 mer oligonucleotide [SEQ IDNO. 16] which was used in multiplex analysis of CpG methylation (site 2)of thep16 5′CpG in combination with a 15-mer and 21-mer primer [SEQ IDNOS 17 and 15, respectively] (correlation coefficient=0.99). Both the 15mer and 21-mer produced a nearly identical linear response as the18-mer. FIG. 3 shows data from three separate experiments.

FIG. 4 shows a schematic diagram that outlines a process for ahigh-throughput methylation analysis. The Ms-SNuPE primer extensionreactions are performed and then the products are directly transferredto membranes, preferably nylon membranes. This allows for a large numberof samples to be analyzed simultaneously in a high-density format. Themembrane is washed and exposed to a phosphorimage cassette forquantitative methylation analysis and eliminate the need forpolyacrylamide gel electrophoresis for data measurement.

FIG. 5 (Panel A) shows results from quantitative analysis of DNAmethylation using the Ms-SNuPE blot transfer technique of FIG. 4. Levelsof DNA methylation in matched normal and tumor colon specimens wereanalyzed in the 5′ promoter region of the p16 gene. The averageethylation of 3 sites in the p16 promoter (FIG. 2) was determined byquantitating the C:T signal ration by phosphorimage analysis. Panel Bshows the results of quantitating the average methylation of 3 CpG sitesusing standard polyacrylamide gel electrophoresis compared to dot blottransfers. The average methylation of the monitored sites in variouscolon specimens is plotted on the graph and shows little differencebetween quantitated values derived from polyacrylamide gelelectrophoresis compared tom the dotblot technique. These data show thefeasibility of using the Ms-SNuPE dotblot procedure for high-throughputdetection and quantitation of DNA methylation changes in cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining DNA methylationpatterns at cytosine sites, comprising the steps of:

(a) obtaining genomic DNA from a DNA sample to be assayed, whereinsources of DNA include, for example, cell lines, blood, sputum, stool,urine, cerebrospinal fluid, paraffin-embedded tissues, histologicalslides and combinations thereof;

(b) reacting the genomic DNA with sodium bisulfite to convertunmethylated cytosine residues to uracil residues while leaving any5-methylcytosine residues unchanged to provide primers specific for thebisulfite-converted genomic sample for top strand or bottom strandmethylation analysis;

(c) performing a PCR amplification procedure using the top strand orbottom strand specific primers;

(d) isolating the PCR amplification products;

(e) performing a primer extension reaction using Ms-SNuPE primers,[³²P]dNTPs and Taq polymerase, wherein the Ms-SNuPE primers comprise afrom about a 15 mer to about a 22 mer length primer that terminatesimmediately 5′ of a single nucleotide to be assayed; and

(f) determining the relative amount of allelic expression of CpGmethylated sites by measuring the incorporation of different ³²P-labeleddNTPs.

Preferably, the [³²P]NTP for top strand analysis is [³²P]dCTP or[³²P]TTP. Preferably, the [³²P]NTP for bottom strand analysis is[³²P]dATP or [³²P]dGTP. Preferably, the isolation step of the PCRproducts uses an electrophoresis technique. Most preferably, theelectrophoresis technique uses an agarose gel.

DNA is isolated by standard techniques for isolating DNA from cellular,tissue or specimen samples. Such standard methods are found in textbookreferences such as Fritsch and Maniatis eds., Molecular Cloning: ALaboratory Manual, 1989.

The bisulfite reaction is performed according to standard techniques.For example and briefly, approximately 1 microgram of genomic DNA(amount of DNA can be less when using micro-dissected DNA specimens) isdenatured for 15 minutes at 45° C. with 2N NaOH followed by incubationwith 0.1M hydroquinone and 3.6M sodium bisulfite (pH 5.0) at 55° C. for12 hours (appropriate range is 4-12 hours). The DNA is then purifiedfrom the reaction mixture using standard (commercially-available) DNAminiprep columns, or other standard techniques for DNA purification arealso appropriate. The purified DNA sample is resuspended in 55microliters of water and 5 microliters of 3N NaOH is added for adesulfonation reaction, preferably performed at 40° C. for 5-10 minutes.The DNA sample is then ethanol-precipitated and washed before beingresuspended in an appropriate volume of water. Bisulfite treatment ofDNA distinguishes methylated from unmethylated cytosines. The presentbisulfite treatment method has advantages because it is quantitative,does not use restriction enzymes, and many CpG sites can be analyzed ineach primer extension reaction by using a multiplex primer strategy.

The PCR amplification step (c) can be performed by standard PCRtechniques, following a manufacturer's instructions. For example,approximately 1-2 microliters of the bisulfite-treated DNA was used as atemplate for strand-specific PCR amplification in a region of interest.In a PCR reaction profile for amplifying a portion of the p16 5′ CpGisland, for example, a procedure of initial denaturation of 94° C. for 3minutes followed by a cycle of 94° C. of 30 seconds, 68° C. for 30seconds, 72° C. for 30 seconds for a total of 30 cycles. The PCRreactions were performed in 25 microliter volumes under conditions of:˜50 ng bisulfite-converted DNA (less for micro dissected samples), 10 mMTris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 0.1% gelatin/ml, 100 μM ofeach of dNTP, 0.5 μM final concentration of each primer and 1 unit ofTaq polymerase. There are many chromatographic techniques that can beused to isolate the PCR amplification products. In one illustrativeprocedure, approximately 10-25 microliters of the amplified PCR productswere loaded onto 2% agarose gels and electrophoresed. The bands werevisualized and isolated using standard get purification procedures.

The primer extension reaction is conducted using standard PCR primerextension techniques but using Ms-SNuPE primers as provided herein.Approximately 10-50 nanograms of purified PCR template is used in eachMs-SNuPE reaction. A typical reaction volume is about 25 microliters andcomprises PCR template (about 10-50 ng), 1×PCR buffer, 1 μM of eachMs-SNuPE primer, 1 μCi of the appropriate ³²P-labeled dNTP (either[³²P]dCTP, [³²P]TTP, [³²P]dATP, [³²P]dGTP or combinations thereof), and1 unit of Taq polymerase. As a general rule, oligonucleotides used inthe primer extension reactions were designed to have annealingtemperatures within 2-3° C. of each other and did not hybridize tosequences that originally contained CpG dinucleotides. The Ms-SNuPEreactions were performed at 95° C. for 1 minute, 50° C. for 2 minutes,and 72° C. for 1 minute. A stop solution (10 microliters) was added tothe mixtures to terminate the reactions. The inventive Ms-SNuPE assayutilizes internal primer(s) which anneal to a PCR-generated template andterminate immediately 5′ of the single nucleotide to be assayed. Asimilar procedure has been used successfully for detection of genemutations Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147,1991) and for quantitation of allele-specific expression (Szabo andMann, Genes Dev. 9:3097-3108, 1995 and Greenwood and Burke, Genome Res.6:336-348, 1996).

There are several techniques that are able to determine the relativeamount of methylation at each CpG site, for example, using a denaturingpolyacrylamide gel to measure ³²P through phosphorimage analysis, ortransfer of Ms-SNuPE reaction products to nylon membranes, or even usingfluorescent probes instead of a ³²P marker. In one method fordetermining the relative amount of methylation at each CpG site,approximately 1-2 microliters of each Ms-SNuPE reaction product waselectrophoresed onto 15% denaturing polyacrylamide gel (7M urea). Thegels were transferred to filter paper and then dried. Phosphorimageanalysis was performed to determine the relative amount of radiolabeledincorporation. An alternative method for determining the relative amountof methylation at individual CpG sites is by a direct transfer of theMs-SNuPE reaction products to nylon membranes. This technique can beused to quantitate an average percent methylation of multiple CpG siteswithout using polyacrylamide gel electrophoresis. High-throughputmethylation analysis was performed by direct transfer of the Ms-SNuPEreactions onto nylon membranes. A total of 100 microliters or 0.4 mMNaOH, 1 mM Na₄P₂O₇ was added to the completed primer extension reactionsinstead of adding stop solution. The mixture was directly transferred tonylon membranes using a dotblot vacuum manifold in a 96 well plateformat. Each vacuum transfer well was washed a total of 4 times with 200microliters of 2×SSC, 1 mM Na₄P₂O₇. The entire membrane was washed in2×SSC, 1 mM Na₄P₂O₇. The radioactivity of each spot on the dried nylonmembrane was quantitated by phosphorimaging analysis.

In the inventive quantitative Ms-SNuPE assay, the relative amount ofallelic expression is quantitated by measuring the incorporation ofdifferent ³²P-labeled dNTPs. FIG. 1 outlines how the assay can beutilized for quantitative methylation analysis. For example, the initialtreatment of genomic DNA with sodium bisulfite causes unmethylatedcytosine to be converted to uracil, which is subsequently replicated asthymine during PCR. Methylcytosine is resistant to deamination and isreplicated as cytosine during amplification. Quantitation of the ratioof methylated versus unmethylated cytosine (C versus T) at the originalCpG sites can be determined by incubating a gel-isolated PCR product,primer(s) and Taq polymerase with either [³²P]dCTP or [³²P]TTP, followedby denaturing polyacrylamide gel electrophoresis and phosphorimageanalysis. In addition, opposite strand (bottom strand) Ms-SNuPE primersare further designed which would incorporate either [³²P]dATP or[³²P]dGTP to assess methylation status depending on which CpG site isanalyzed.

Ms-SNuPE Primers

The present invention further provides a Ms-SNuPE primer sequencedesigned to anneal to and terminate immediately 5′ of a desired cytosinecodon in the CpG target site and that is located 5′ upstream from a CpGisland and are frequently hypermethylated in promoter regions of somaticgenes in malignant tissue. Preferably, the Ms-SNuPE primer sequencecomprises a sequence of at least fifteen bases having a sequenceselected from the group consisting of GaL1 [SEQ ID NO. 1], GaL2 [SEQ IDNO. 2], GaL4 [SEQ ID NO. 3], HuN1 [SEQ ID NO. 5], HuN2 [SEQ ID NO. 6],HuN3 [SEQ ID NO. 7], HuN4 [SEQ ID NO. 8], HuN5 [SEQ ID NO. 8], HuN6 [SEQID NO. 9], CaS1 [SEQ ID NO. 10], CaS2 [SEQ ID NO. 11], CaS4 [SEQ ID NO.12], and combinations thereof. The present invention further provides amethod for obtaining a Ms-SNuPE primer sequence, comprising finding ahypermethylated CpG island in a somatic gene from a malignant tissue orcell culture, determining the sequence located immediately 5′ upstreamfrom the hypermethylated CpG island, and isolating a 15 to 25 mersequence 5′ upstream from the hypermethylated CpG island for use as aMs-SNuPE primer. The present invention further provides a Ms-SNuPEprimer comprising a 15 to 25 mer oligonucleotide sequence obtained bythe process comprising, (a) identifying hypermethylated CpG islands asomatic gene from a malignant tissue or cell culture source, (b)determining the sequence located immediately 5′ upstream from thehypermethylated CpG island, and (c) isolating at least a 15 mer sequence5′ upstream from the hypermethylated CpG island for use as a Ms-SNuPEprimer. Preferably the Ms-SNuPE primer sequence is from about 15 toabout 25 base pairs in length.

The ability to detect methylation changes associated with oncogenictransformation is of critical importance in understanding how DNAmethylation may contribute to tumorigenesis. Regions of DNA that havetumor-specific methylation alterations can be accomplished using avariety of techniques. This will permit rapid methylation analysis ofspecific CpG sites using the inventive quantitative Ms-SNuPE primerprocess. For example, techniques such as restriction landmark genomicscanning (RLGS) (Hatada et al., Proc. Natl. Acad. Sci. USA 88:9523-9527,1995), methylation-sensitive-representational difference analysis(MS-RDA) (Ushijima et al., Proc. Natl. Acad. Sci. USA 94:2284-2289,1997) and methylation-sensitive arbitrarily primed PCR (AP-PCR)(Gonzalgo et al., Cancer Res. 57: 594-599, 1997) can be used foridentifying and characterizing methylation differences between genomes.

Briefly, sequence determinations of regions of DNA that showtumor-specific methylation changes can be performed using standardtechniques, such as those procedures described in textbook referencessuch as Fritsch and Maniatis eds., Molecular Cloning: A LaboratoryManual, 1989. Additionally, commercially available kits or automated DNAsequencing systems can be utilized. Once specific regions of DNA havebeen identified by using such techniques, the Ms-SNuPE primers can beapplied for rapidly screening the most important CpG sites that areinvolved with the specific methylation changes associated with a cancerphenotype.

EXAMPLE 1

This example illustrates a quantitative methylation analysis of threetop strand sites in a 5′ CpG island of p16 in various DNA samples usingthe inventive method. The top panel provides the locations of threesites analyzed (numbered 1, 2 and 3) relative to the putativetranscriptional start sites (vertical arrows pointing upwards) and theexon 1α coding domain. The PCR primers used for top strand amplificationof the 5′ region of p16 (which includes putative transcriptional startsites) were 5′-GTA GGT GGG GAG GAG TTT AGT T-3′ [SEQ ID NO. 13] and5′-TCT AAT AAC CAACCA ACC CCT CC-3′ [SEQ ID NO. 14]. The reactions wereperformed in 25 μl total volume under the conditions of 50 ngbisulfite-treated DNA, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl,0.1% gelatin/ml, 100 μM of each dNTP, 0.5 μM final concentration of eachprimer and 1 U of Taq polymerase (Boehinger Mannheim, Indianapolis,Ind.). The reactions were hot-started using a 1:1 mixture ofTaq/TaqStart antibody (Clontech, Palo Alto, Calif.).

An initial denaturation of 94° C. for 3 minutes was followed by 94° C.for 30 sec. 68° C. for 30 sec, 72° C. for 30 sec for a total of 35cycles. The PCR products were separated by electrophoresis on 2% agarosegels and the bands were isolated using a Qiaquick™ gel extraction kit(Qiagen, Santa Clarita, Calif.).

The Ms-SNuPE reaction was performed in a 25 ml reaction volume with10-50 ng of PCR template incubated in a final concentration of 1×PCRbuffer, 1 μM of each Ms-SNuPE primer, 1 μCi of either [³²P]dCTP or[³²P]TTP and 1 U of Taq polymerase. The primer extensions were alsohot-started using a 1: mixture of Taq/TaqStart antibody. The primersused for the Ms-SNuPE analysis were: site 1 5′-TTT TTT TGT TTG GAA AGATAT-3′ [SEQ ID NO. 15]; site 2 5′-TTT TAG GGG TGT TAT ATT-3′ [SEQ ID NO.16]; site 3 5-TTT GAG GGA TAG GGT-3′ [SEQ ID NO. 17]. The conditions forthe primer extension reactions were 95° C. for 1 minute, 50° C. for 2minutes and 70° C. for 1 minute. A stop solution (10 μl) was added tothe reaction mixtures and the samples were loaded onto 15% denaturingpolyacrylamide gels (7 M urea). Radioactivity of the bands wasquantitated by phosphorimaging analysis. The control sets included “M”PCR product amplified from a plasmid containing bisulfide-specificmethylated sequence; “U” PCR product amplified from a plasmid containingbisulfite-specific unmethylated sequence; and “mix” a 50:50 mixture ofmethylated and unmethylated PCR-amplified plasmid sequences. The DNAsamples analyzed included T24 and J82 bladder cancer cell lines; wbc(white blood cell), melanoma (primary melanoma tumor tissue sample), andbladder (primary bladder tumor tissue sample). The tissue samples weremicro dissected from paraffin-embedded tumor material. The grid at thebottom of the lower panel shows the ratio of methylated (C) versusunmethylated (T) bands at each site based upon phosphorimagequantitation.

These data (FIG. 2) show the ability of the inventive assay to detectaltered patterns of methylation.

EXAMPLE 2

This example illustrates a mixing experiment showing a linear responseof the inventive Ms-SNuPE assay for detection of cytosine methylation. AT24 bladder cancer cell line DNA (predominantly methylated) was added inincreasing amounts to a J82 bladder cancer cell line DNA (predominantlyunmethylated). FIG. 3 shows data from an 18 mer oligonucleotide [SEQ IDNO. 16] which was used in multiplex analysis of CpG methylation (site 2)of the p16 5′CpG in combination with a 15-mer and 21-mer primer [SEQ IDNOS 17 and 15, respectively] (correlation coefficient=0.99). Both the 15mer and 21-mer produced a nearly identical linear response as the18-mer. FIG. 3 shows data from three separate experiments. Differentialspecific activity and incorporation efficiency of each [³²P]dNTP wascontrolled for by using a 50:50 mixture of bisulfite-specific methylatedversus unmethylated PCR template for analysis.

EXAMPLE 3

This example provides a summary of DNA regions for which Ms-SNuPEprimers can be designed and the inventive method applied for aquantitative detection of abnormal DNA methylation in cancer cells. Thesequences are listed according to name, size and frequency ofhypermethylation in the corresponding cell line or primary tumor.

hyper- hyper- methylated methylated methylated frag- size in colon incolon in bladder ment (bp) cell line cancer cancer comments GaL1 530 7/7(100%) 3/7 (42%) 3/7 (42%) GC content (0.6), observed/ expected CpG(0.63) GaL2 308 7/7 (100%) 4/5 (80%) 6/7 (85%) GC content (0.6),observed/ expected CpG (0.6) GaL4 177 7/7 (100%) 1/2 (50%) 3/4 (75%) GCcontent (0.59), observed/ expected CpG (0.50) CaS1 215 4/7 (57%) 0/5(0%) 2/7 (28%) GC content (0.55), observed/ expected CpG (0.78) CaS2 2204/7 (57%) 3/5 (60%) 3/7 (42%) GC content (0.54), observed/ expected CpG(0.74) CaS4 196 6/7 (85%) 0/5 (0%) 1/7 (14%) GC content (0.64),observed/ expected CpG (0.84) HuN1 148 7/7 (100%) 3/5 (60%) 3/7 (42%) GCcontent (0.54), observed/ expected CpG (0.99) HuN2 384 7/7 (100%) 4/5(80%) 2/7 (28%) GC content (0.6), observed/ expected CpG (0.62) HuN3 1786/7 (85%) 4/5 (80%) 3/7 (42%) GC content (0.53), observed/ expected CpG(0.97) HuN4 359 7/7 (100%) 3/5 (60%) 4/7 (57%) GC content (0.51),observed/ expected CpG (0.47) HuN5 251 7/7 (100%) 2/5 (40%) 5/7 (71%) GCcontent (0.63), observed/ expected CpG (0.77) HuN6 145 6/7 (85%) 3/4(75%) 1/2 (50%) GC content (0.55), observed/ expected CpG (0.47)

17 530 nucleic acid single unknown GaL1 not provided 1 CCCGCGACCTAAGCCAGCGA CTTACCACGT TAGTCAGCTA AGAAGTGGCA 50 GAGCTGGGAT TCGAACCTATAAAGAACTCT GAAGCCTGGG TATTTTTACA 100 TGACACTTTA CATAATGCGC CACGGGGTAGTCGGAGGGGG AGGTCCATCT 150 CCCTTTCCCT TGCTGTCCAT CTCCACAGAA AAGAAGCAAGTGGAGGACAG 200 GAGCCAGAAA GTCATCTGGC CGCGGATCAT TCCGGAGTGA CCCCCGCCGC250 CACCACTCGC ATAGTCCGCT TATGGCGGGA GGGCACCTCA GAGATTCTCA 300CAGGGGCTGT GCGGCCAGAA CCAGAAGTGC AAAGCACCGT TAGCGACTCT 350 ATCGCCCCCTGCCGCCTGTG GCGCCCAGTC CGAAGCTGCT GTTTTCAGGA 400 GGGCTAGTGG GCTAAGAAAAGAGCTCACCG ACTGACTGCC CAACAGCTGT 450 TGCGAGCCAG TGCTAGGCTG CAGACAGCCTTGCCAAATGT GGTGACATAA 500 GCGGGAGGGG GGAACATTTA GAGAGCCCTA 530 308nucleic acid single unknown GaL2 not provided 2 CTAGGGTAGG CTGGTCTGTGCTGGATACGC GTGTTCTTCT GCGGAGTTAA 50 AGGGTCGGGG ACGGGGGTTC TGGACTTACCAGAGCAATTC CAGCCGGTGG 100 GCGTTTGACA GCCACTTAAG GAGGTAGGGA AAGCGAGCTTCACCGGGCGG 150 GCTACGATGA GTAGCATGAC GGGCAGCAGC AGCAGCAGCC AGCAAAAGCC200 TAGCAAAGTG TCCAGCTGCT GCACTGCCGC GGGGACTCCC ACATCACCAT 250GACTAGTTGT GCAACTCTGC AGCAGAAACG GCTTCCGAGG AACACAGGAT 300 CGCGGGGG 308177 nucleic acid single unknown GaL4 not provided 3 GCTTCCTTTTTCTCGGCTTT CCTCACTATC CTCTCCCTGT TCGAGAGTAT 50 CTCCACCAGC ACCGAGCCTCACACGGGCTG TGCCTCCATC TTTGGAATGC 100 CTACCCTTCT TTCTTGCGAA GCCCCTCCCAGGGCCAGCCC TTGTGCACCG 150 GCTCAAGGGG ACTGCTCTCC TGCCTCG 177 148 nucleicacid single unknown HuN1 not provided 4 TTGCGCCGAT CGTCAAGAAC CTCTCATCCCTGGCAGCAGC AAAGCCAATA 50 TATTTCCATT TCTTATTTCA GTTTGCCACC AAAACAAAGCTGCGCGCGGC 100 TGAGGGCAGG AAGGCGCTGA GACCGACCGA GAAGAAGGGA CGTCCCGG 148384 nucleic acid single unknown HuN2 primer not provided 5 CAGGCCCGCCGAGACTCCAC TCCAACTACC AGGAAATTTC CCGTGGAGCT 50 TCAATTCCTG GGACCCTCCTACTGCGGGGA GAGTGGTTTC CCTGCCCCAC 100 ACCATGCCCT AGGCCCGAGT CTGCGGCTCTTGGGGGATCT CTCCGAGCTC 150 CGACACCGTG TTCGGACCGG GTGCGCCCTG CCGCTGGGGCTCAAGCCTGC 200 AGGCGTGAGA ACCGGGGGAC TCTCTATGGC ACCAAGAGCT TCACCGTGAG250 CGTAGGCAGA AGCTTCGCTT TGATCCTAGG GCTTACAAAG TCCTCCTTTG 300GCTGCCCATG ATGGTAAAAG GGCAGTTGCT CACAAAGCGC GAGTGTGTGT 350 GCCAGACAGTGTAAATGAGT GTTGGGACCG GCGT 384 178 nucleic acid single unknown HuN3 notprovided 6 GGGTCCGTTC GTGAATGCAT GAGCAGGGTG TGAGCGCCAG GGGGTTACAC 50TTCTCACGGG TTAAAACCCA GACAACTTCA CGAGGGAACC ACGTGCCATT 100 TTAACAGCGTACGGTCGGGA TCGTGGGACG TCATTAAACG GAGTGGGTTG 150 AGTATGTGAC TCTGTCACCCATTTTCTG 178 359 nucleic acid single unknown HuN4 primer not provided 7CCCCGCGGGG CAGAATCCAA GTGAGTCAGA CACATTGCTC CCTCCCTGCT 50 GCTGCCAGTCCATCTCTTTG CCAACAAACC TGCTTAAAAT GCCAAAGCTG 100 GTCCAAAGTT TCAGGAAAACAACTTCCGCC AGAGGGCACG TAGAGGGCAC 150 AGATGCTATA GATGCTTCTC TGACAAACACTCCTGACCCC CTTGACAGAT 200 TGGAAAATAC ATGGTTCAGA AAGGGTGAGA GATTTCAACTTGAGAAGTGA 250 AACTAGGAAA AGATGGAAGG TGTCCGGATT TCTAGCTCAA GTCCACACAC300 TGCTTCTGCT GCGGTGACTA AATCGTGGCT GTGTTCTCAT CACCTGCCTC 350 GCGGCGCGC359 251 nucleic acid single unknown HuN5 primer not provided 8GGCGGGCCTG GGCACCGCGG AGGGGGGGCT TTTCTGCGCC CGGCGAAGCG 50 TGGAACTTGCGCCCTGAGGC AGCGCGGCGA GACCAGTCCA GAGACCGGGG 100 CGAGCCTCCT CAGGATTCCTCGCCCCAGTG CAGATGCTGT GAGCTTAGAC 150 GAGGACAGGG CATGGCACTC GGCTTGGCCCGTAGTGGACG GTGTTTTTGC 200 AGTCATGAAC CCAAACGCCG CAAACCTTGA CCGTTTCCCCACCCGTGTTG T 251 145 nucleic acid single unknown HuN6 primer notprovided 9 TGAGAGCAGC ATCCTCCCCT GCGTGTGGTT CTCTAACTTA CCTCCTGTAT 50GGGGTCTGCG GACCCAGCAC ACCTCCCGGG CCCCCAAAAA ATTCCAGCTC 100 AAGAGCCCTAAAAATCCTTA CCCTGNNAAA GTTTGAGCTT CTCCC 145 215 nucleic acid singleunknown CaS1 primer not provided 10 ACGCCGGCCA CAGTTCTTCA GTGAAACGCTTCACTCTCTG GTCATAGAGG 50 TAGGAAACTA TAGCTGTCCC AACTAAATGT CAGGACGAATTAGCCCAGCT 100 GGTCACGCTC ACAGTCACCG CCTCCACCAG ACTGAGCGAC CCTCCCAACG150 GGGTTTGCCG TGTTGGGAGG ACAGCGGAGT TTCGTTGCTG TGTCAATTTG 200TGTAGACGCG GCTGC 215 220 base pairs nucleic acid single unknown CaS2primer not provided 11 CTGCTCTCTT CTCTTCTTTT CCCCTTTCCT CTCCTCTCCCTTTCCTCAGG 50 TCACAGCGGA GTGAATCAGC TCGGTGGTGT CTTTGTCAAC GGGCGGCCAC 100TGCCGGACTC CACCCGGCAG AAGATTGTAG AGCTAGCTCA CAGCGGGGCC 150 CGGCCGTGCGACATTTCCCG AATTCTGCAG GTGATCCTCC CGGCGCCGCC 200 CCACTCGCCG CCCCCGCGGC220 196 nucleic acid single unknown CaS4 primer not provided 12GGGCGGCACG GAGGGAGTCA GGAGTGAGCC CGAAGATGGA GAGAAGTCGA 50 TTCGCCCAGAGAACGCAAGA CGGTGGATCA GAGATGAGTC CCAGGAACCT 100 CAGAGAGCGA GGCTGACAGGCCCGGGGAGA GGACCGGGCA GGGACAAACC 150 AGCGGACAGA GCAGAGCGCG AAATGGTTGAGACCGGGAAG CGACCT 196 22 nucleic acid single unknown Ms-SNuPE primerfrom p16 promoter region not provided 13 GTA GGT GGG GAG GAG TTT AGT T22 23 nucleic acid single unknown Ms-SNuPE primer from p16 promoterregion not provided 14 TCT AAT AAC CAA CCA ACC CCT CC 23 21 nucleic acidsingle unknown Ms-SNuPE primer from p16 promoter region not provided 15TTT TTT TGT TTG GAA AGA TAT 21 18 nucleic acid single unknown Ms-SNuPEprimer from p16 promoter region not provided 16 TTT TAG GGG TGT TAT ATT18 15 nucleic acid single unknown Ms-SNuPE primer from p16 promoterregion not provided 17 TTT GAG GGA TAG GGT 15

We claim:
 1. A method for determining DNA methylation status at acytosine residue of a CpG sequence, comprising the steps of: (a)obtaining genomic DNA from a DNA sample to be assayed; (b) reacting thegenomic DNA with sodium bisulfite to convert unmethylated cytosineresidues to uracil residues while leaving any 5-methylcytosine residuesunchanged to create an exposed bisulfite-converted DNA sample havingbinding sites for primers specific for the bisulfite-converted DNAsample; (c) performing a PCR amplification procedure using top strand orbottom strand specific primers; (d) isolating the PCR amplificationproducts; (e) performing a primer extension reaction using a Ms-SNuPEprimer, (³²P) dNTPs and Taq polymerase, wherein the Ms-SNuPE primercomprises from about a 15 mer to about a 22 mer length primer sequencethat is complementary to the bisulfite-converted DNA sample andterminates immediately 5′ of the cytosine residue of the CpG sequence tobe assayed; and (f) determining the methylation status at the cytosineresidue of the CpG sequence by measuring the incorporation of different³²P-labeled dNTPs.
 2. The method of claim 1 wherein the [³²P]dNTP fortop strand analysis is [³²P]dCTP or [³²P]TTP.
 3. The method of claim 1wherein the [³²P]dNTP for bottom strand analysis is [³²P]dATP or[³²P]dGTP.
 4. The method of claim 1 wherein the isolation step of thePCR products uses an electrophoresis technique.
 5. The method of claim 4wherein the electrophoresis technique uses an agarose gel.
 6. The methodof claim 1 wherein the Ms-SNuPE primer comprises a sequence of at leastfifteen but no more than twenty five nucleotides of a sequence selectedfrom the group consisting of GaL1 (SEQ ID NO: 1), GaL2 (SEQ ID NO: 2),GaL4 (SEQ ID NO: 3), HuN1 (SEQ ID NO: 4), HuN2 (SEQ ID NO: 5), HuN3 (SEQID NO: 6), HuN4 (SEQ ID NO: 7), HuN5 (SEQ ID NO: 8), HuN6 (SEQ ID NO:9), CaS1 (SEQ ID NO: 10), CaS2 (SEQ ID NO: 11), CaS4 (SEQ ID NO: 12). 7.A Ms-SNuPE primer that terminates immediately 5′ upstream of a cytosineresidue in a CpG sequence of a CpG island that is frequentlyhypermethylated in promoter regions of somatic genes in malignanttissue, wherein said Ms-SNuPE primer comprises an oligonucleotideconsisting of at least 15 contiguous nucleotides of a gene sequencelocated immediately 5′ upstream from the CpG sequence.
 8. The Ms-SNuPEprimer sequence of claim 7 wherein the primer sequence is from about 15to about 25 nucleotides in length and selected from the group consistingof GaL1 (SEQ ID NO: 1), GaL2 (SEQ ID NO: 2), GaL4 (SEQ ID NO: 3), HuN1(SEQ ID NO: 4), HuN2 (SEQ ID NO: 5), HuN3 (SEQ ID NO: 6), HuN4 (SEQ IDNO: 7), HuN5 (SEQ,ID NO: 8), HuN6 (SEQ ID NO: 9), CaS1 (SEQ ID NO: 10),CaS2 (SEQ ID NO: 11), CaS4 (SEQ ID NO: 12).
 9. A method for obtaining aMs-SNuPE primer sequence that terminates immediately 5′ of a cytosineresidue in a CpG sequence of a CpG island, comprising finding ahypermethylated CpG sequence in a CpG island in a somatic gene from amalignant tissue or cell culture, determining the sequence locatedimmediately 5′ upstream from the hypermethylated CpG sequence, andsynthesizing, based at least in part on using the sequence locatedimmediately 5′ upstream from the hypermethylated CpG sequence as atemplate, a Ms-SNuPE primer comprising a 15 to 25 nucleotide sequenceimmediately 5′ upstream from the hypermethylated CpG sequence.
 10. AMs-SNuPE primer comprising a 15 to 25 mer oligonucleotide sequenceobtained by the process comprising finding a hypermethylated CpGsequence in a CpG island in a somatic gene from a malignant tissue orcell culture, determining the sequence located immediately 5′ upstreamfrom the hypermethylated CpG sequence, and synthesizing, based at leastin part on using the sequence located immediately 5′ upstream from thehypermethylated CpG sequence as a template, a Ms-SNuPE primer comprisinga 15 to 25 nucleotide sequence immediately 5′ upstream from thehypermethylated CpG sequence, wherein said primer terminates immediately5′ upstream from the hypermethylated CpG sequence.
 11. The method ofclaim 1, wherein performing a primer extension reaction comprisessimultaneous use of a plurality of unique MS-SNuPE primers, and whereineach primer comprises from about a 15 mer to about a 22 mer lengthprimer sequence that is complementary to the bisulfite-converted DNAsample and terminates immediately 5′ of one of a plurality of unique CpGsequences, whereby the relative methylation status of the plurality ofunique CpG sequences can be simultaneously determined.